Multiparametric method for assessing immune system status

ABSTRACT

The invention provides a multiparametric method of assessing the reaction of a patient&#39;s immune system to a test subject. The invention compares a patient sample reacted with a test sample and a third party sample and combines the assessments of the multiple parameters to correlate the test reaction with a clinical event.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Patent application Ser. No.15/432,858, filed on Feb. 14, 2017, issued as U.S. Pat. No. 9,772,326,which is a continuation of U.S. Patent application Ser. No. 14/850,628,filed on Sep. 10, 2015, issued as U.S. Pat. No. 9,606,109, which is acontinuation of U.S. Patent application Ser. No. 14/278,306, filed May15, 2014, abandoned, which is a divisional of U.S. Patent applicationSer. No. 13/847,252, filed Mar. 19, 2013, issued as U.S. Pat. No.8,759,016, which is a divisional of U.S. Patent application Ser. No.11/447,213, filed Jun. 5, 2006, issued as U.S. Pat. No. 8,426,146, whichin turn claims the benefit of U.S. Provisional Application No.60/687,403, filed Jun. 3, 2005. The prior applications are allincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

The commonest early problems seen in transplant patients includerejection or side effects of anti-rejection drugs. This is observed inabout half of all subjects. These complications remain a source ofconcern throughout the post-transplant course. Minimization ofanti-rejection medications to alleviate side-effects presents a dilemmabecause it is attended by rejection in up to about one third of allpatients. Moreover, as long as anti-rejection medications are used,transplant patients continue to experience a lifelong risk oflife-threatening infections and cancers.

These complications result from the interplay of several factors, suchas infectious agent or virus, transplanted tissue, host immune system,and anti-rejection drugs. The net effect that is seen as a clinicalmanifestation is therefore the result of multiple biochemical ormolecular pathways that can be affected by these factors alone or incombination, to varying degrees. Current technology to assess suchfactors operates linearly, measuring one or only a few at a time. Suchtechnology does not take into account the complexity of immuneinteractions. Thus, there is a need for the ability to monitor suchmultiple interrelated events and to deliver integrated output forclinical decision making.

BRIEF SUMMARY

The invention provides a multiparametric method of assessing thereaction of a patient's immune system to a test subject, who may be anorgan donor or someone who is immunologically similar to the patient.The method includes: a) obtaining a first sample, a second sample, and athird sample from the patient, wherein the first sample and the secondsample comprise lymphocytes, peripheral blood mononuclear cells (PBMCs),or a mixture of lymphocytes and peripheral blood mononuclear cells, andlabeling the cells with a first marker or first set of markers, b)obtaining a test sample from a test subject, wherein the test subject isan individual other than the patient, and wherein the test samplecomprises cells similar to the cells within the first sample obtainedfrom the patient, and labeling the cells with a marker or set ofmarkers, c) obtaining a third party sample from a third party, whereinthe third party is an individual other than the patient and other thanthe test subject, and wherein the third party is immunologicallydissimilar to the patient and the test subject, and wherein the thirdparty sample comprises cells similar to the cells within the secondsample obtained from the patient, and optionally labeling the cells witha marker or a set of markers, d) introducing the first sample obtainedfrom the patient and the test sample obtained from the test subject intoa first vessel under conditions sufficient for the cells within thefirst sample and the test sample to react, e) introducing the secondsample obtained from the patient and the third party sample obtainedfrom the third party into a second vessel under conditions sufficientfor the cells within the second sample and the third party sample toreact, wherein the second vessel is other than the first vessel, f)introducing the third sample obtained from the patient into a thirdvessel and subjecting it to similar conditions as the first and secondvessel to serve as a control, g) measuring multiple parameters of thereaction in the first vessel to obtain a test measurement for eachparameter, h) measuring multiple parameters of the reaction in thesecond vessel to obtain a third party measurement for each parameter, i)comparing each test measurement of a parameter to each third partymeasurement of the same parameter, whereby the test measurement isexpressed as a fraction of the third party measurement to provide anassessment of the parameter, and j) combining the assessments of themultiple parameters to correlate the test reaction with a clinicalevent.

The invention also provides a multiparametric method of assessing thereaction of a patient's immune system to a test stimulant. The methodincludes: a) obtaining a first sample, and a second sample from thepatient, wherein the first sample and the second sample compriselymphocytes, peripheral blood mononuclear cells (PBMCs), or a mixture oflymphocytes and peripheral blood mononuclear cells, and optionallylabeling the cells with a first marker or set of markers, b) subjectingthe patient's first sample to stimulant in a first vessel underconditions sufficient for the patient's cells to react with the antigen,c) placing the patient's second sample into a second vessel underconditions similar to those of the first vessel to serve as a control,d) measuring multiple parameters of the reaction in the first vessel toobtain a test measurement for each parameter, e) measuring multipleparameters of the reaction in the second vessel to obtain a controlmeasurement for each parameter, f) comparing each test measurement of aparameter to each control measurement of the same parameter, whereby thetest measurement is expressed as a fraction of the control measurementto provide an assessment of the parameter, and g) combining theassessments of the multiple parameters to correlate the test reactionwith a clinical event.

The invention further provides a multiparametric method of assessing thereaction of a test subject's immune system to a patient. The methodcomprises: a) obtaining a first sample, a second sample, and a thirdsample from the test subject, wherein the test subject isimmunologically similar to the patient, and the first sample, secondsample, and third sample comprise lymphocytes, peripheral bloodmononuclear cells (PBMCs), or a mixture of lymphocytes and peripheralblood mononuclear cells, and labeling the cells with a marker or set ofmarkers, b) obtaining a patient sample from the patient, wherein thepatient's sample comprises cells similar to the cells within the firstsample obtained from the patient, and optionally labeling the cells witha marker or set of markers, c) obtaining a third party sample from athird party, wherein the third party is an individual other than thepatient and other than the test subject, and wherein the third party isimmunologically dissimilar to both the patient and the test subject, andwherein the third party sample comprises cells similar to the cellswithin the second sample obtained from the patient, and optionallylabeling the cells with a marker or a set of markers, d) introducing thefirst sample obtained from the test subject and the patient's sampleobtained from the patient into a first vessel under conditionssufficient for the cells within the first sample and the patient'ssample to react, e) introducing the second sample obtained from the testsubject and the third party sample obtained from the third party into asecond vessel under conditions sufficient for the cells within thesecond sample and the third party sample to react, wherein the secondvessel is other than the first vessel, f) introducing the third sampleobtained from the test subject into a third vessel and subjecting it tosimilar conditions as the first and second vessel to serve as a control,g) measuring multiple parameters of the reaction in the first vessel toobtain a test measurement for each parameter, h) measuring multipleparameters of the reaction in the second vessel to obtain a third partymeasurement for each parameter, i) comparing each test measurement of aparameter to each third party measurement of the same parameter, wherebythe test measurement is expressed as a fraction of the third partymeasurement to provide an assessment of the parameter, and j) combiningthe assessments of the multiple parameters to correlate the testreaction with a clinical event.

The inventive methods and reagents can permit customized drug deliverytargeted to the existing balance between the host immune system and itsmodulation by transplanted organ or infectious agent. The invention alsocan allow transplant patients to experience the benefits ofanti-rejection drugs while minimizing the risk of side effects. Theseand other benefits, as well as additional inventive features, will beapparent from a review of the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a multiparametric immune system monitoring assay.The method measures multiple events at a single cell level, in targetblood cells using flow cytometric techniques. These cells areparticipants in all immune responses toward a diverse range of stimulisuch as transplanted (foreign) tissue, infectious agents such as virusesand bacteria, and drugs that affect the immune system as their intended(anti-rejection medications, medications for autoimmune states, andmedications for allergies) or unintended (cancer drugs) target. Rarecellular subpopulations are identified by multiple identifiers,subjected to a simulated transplanted environment in a vessel, and aresubsequently analyzed.

In one embodiment, the invention provides a multiparametric method ofassessing the reaction of a patient's immune system to a test subject.The method includes: a) obtaining a first sample, a second sample, and athird sample from the patient, wherein the first sample and the secondsample comprise lymphocytes, peripheral blood mononuclear cells (PBMCs),or a mixture of lymphocytes and peripheral blood mononuclear cells, andlabeling the cells with a first marker or first set of markers, b)obtaining a test sample from a test subject, wherein the test subject isan individual other than the patient, and wherein the test samplecomprises cells similar to the cells within the first sample obtainedfrom the patient, and labeling the cells with a marker or set ofmarkers, c) obtaining a third party sample from a third party, whereinthe third party is an individual other than the patient and other thanthe test subject, and wherein the third party is immunologicallydissimilar to the patient and the test subject, and wherein the thirdparty sample comprises cells similar to the cells within the secondsample obtained from the patient, and optionally labeling the cells witha marker or a set of markers, d) introducing the first sample obtainedfrom the patient and the test sample obtained from the test subject intoa first vessel under conditions sufficient for the cells within thefirst sample and the test sample to react, e) introducing the secondsample obtained from the patient and the third party sample obtainedfrom the third party into a second vessel under conditions sufficientfor the cells within the second sample and the third party sample toreact, wherein the second vessel is other than the first vessel, f)introducing the third sample obtained from the patient into a thirdvessel, which is other than the first and second vessel, and subjectingit to similar conditions as the first and second vessel to serve as acontrol, g) measuring multiple parameters of the reaction in the firstvessel to obtain a test measurement for each parameter, h) measuringmultiple parameters of the reaction in the second vessel to obtain athird party measurement for each parameter, i) comparing each testmeasurement of a parameter to each third party measurement of the sameparameter, whereby the test measurement is expressed as a fraction ofthe third party measurement to provide an assessment of the parameter,and j) combining the assessments of the multiple parameters to correlatethe test reaction with a clinical event.

Therefore, the invention provides a method for taking multiparametricmeasurements of the patient's sample, the test sample and the thirdparty sample. Multiple parameters in each of these different samples canbe assessed and therefore the interaction of the cells comprising thepatient's immune system can be assessed.

The patient can be veterinary patient (e.g., small or large domesticanimal) but more typically is a human child or adult. The patient can bean organ transplant candidate, or recipient or a non-organ transplantcandidate or recipient. The patient can be undergoing treatment with animmunosuppressive regimen for a variety of conditions such as actual orsuspected organ rejection, an autoimmune condition, or an allergicreaction. The patient can, for example, be undergoing treatment of aninfectious disease.

A sample (e.g., first sample, second sample, third sample, etc.) can beany tissue drawn from the patient, test subject, or a third party.Typically, the sample will include white blood cells, preferablylymphocytes and/or PBMCs. Desirably, the first, second, and thirdsamples drawn from the patient are substantially identical, and mostpreferably, the first second, and third samples are separate aliquotsobtained from a common initial sample from the patient.

The test subject can be an organ donor, a candidate organ donor, anallergen, or other immunologically stimulating agent. When the testsubject is an organ donor the donor can be an actual donor or acandidate donor. Moreover, the test subject can be a surrogate organdonor, if blood or tissue is unavailable from an actual donor orcandidate donor. A surrogate donor desirably is immunologically similarat the class 1 and 2 major histocompatability loci to the actual donor.The donor should be immunologically compatible with the patient (asdetermined by similarity at the major histocompatibility antigen classes1 and 2), can be living or deceased and the test sample obtainedtherefrom can be lymphocytes and other peripheral mononuclear bloodcells obtained from blood taken from the donor, or tissue obtained fromthe donor's spleen. Preferably, donor cells are live, functioning cells,rather than cells that have been irradiated. When the test subject is animmunologically stimulating agent, other than that obtained from adonor, the test sample can additionally contain antigens such asautoantigens, peptides representing viral or bacterial antigens,antigen-attached to tetramers, and combinations thereof.

The third party can be a live subject or an allergen, or otherimmunologically stimulating agent. Where the third party is a livesubject, (s)he should be immunologically dissimilar to the patient andthe test subject, for example, as determined by similarity at the majorhistocompatibility antigen classes 1 and 2. The third party sample caninclude lymphocytes and other peripheral mononuclear blood cells ortissue obtained from the third party's spleen. When the third party isan immunologically stimulating agent, the sample can additionallycontain antigens such as autoantigens, peptides representing viral orbacterial antigens, antigen-attached to tetramers, and combinationsthereof.

In some embodiments, the cells of the test sample and the third partysample are labeled with a marker or set of markers before placing themin the vessel with the patient's samples to react. Such a marker or setof markers can be a specific marker antibody or marker dye thatidentifies the labeled cells. The marker can either be on the surface ofthe cells or within the cells, and it should stay with such cells forthe life of the reaction. Alternatively, or in addition, the first,second, and third samples from the patients are similarly labeled. Ininstances in which both the cells from the patient and the cells withwhich the cells from the patient are to react (i.e., the test sample andthe third party sample) are labeled, different markers or sets ofmarkers preferably are used so as to distinguish the cells from thepatient from those either from the test subject or third party.

In accordance with the present invention, the first and second samplesfrom the patient are introduced into vessels (i.e., first and secondvessels) with samples from the test subject and third party,respectively, under conditions sufficient for the cells in the vesselsto react. Any suitable conditions can be used, but typically the cellsare maintained in the vessel at about 37° C. for varying degrees of timeso that they can react. Some reactions could be almost immediate, andthe cells in the reaction could be assessed after a time of one or a fewminutes. More typically, the period of time for the reaction is at leastabout 4 hours, or at least about 12 hours or overnight, and the reactionalso can be run for one or several days. Preferably, the reaction is runfor about 24 hours. As noted, the third sample from the patient ismaintained under similar conditions as the test and third partyreactions, so as to serve as a control for the culture conditions.

After the period of time set for the reaction of the cells from the testsubject and the third party, the method involves measuring the multipleparameters. One type of the multiple parameters can be a physicalcharacteristic of the cell. Physical characteristics can include cellsize, complexity, and the degree of aggregation of the cells. In someembodiments, confounding cellular aggregates can be excluded fromanalysis and the parameters of single cells measured. In otherembodiments, however, the degree of aggregation is itself a parameterthat is assessed.

Another type of parameter that can be measured in the context of theinventive method is the cell type. Suitable cell types includelymphocytes and antigen-presenting cells, such as monocytes, dendriticcells, B-cells, macrophages, and the like. Cell subtype is anotherparameter that can be determined. Examples of cell subtypes includenatural killer, T-helper and T-cytotoxic lymphocytes, as well asimmunosuppressive or pro-inflammatory cells. Other cell types andsubtypes can be used as parameters as desired.

Another type of parameter that can be measured in the context of theinventive method is the cells' functional state. Examples of functionalstates that can be measured include death, apoptosis, proliferation,cytokine producing, a particular stage in the cell cycle (e.g., G₀, G₁,G₂, etc.), for example using propidium iodide, degree of activity andwhether the cells have memory or are naïve. Examples of the degree ofcellular activity are highly activated, activated, and inactive. Typesof memory include but are not limited to effector and central memory.

Another example of a cell function that can serve as a parameter to bemeasured in the context of the present invention is whether the cellproduces one or more proteins such as cytokines. Cytokines can beinflammatory cytokines such as interferon gamma (IFN-gamma), tumornecrosis factor alpha (TNF-alpha), interleukin-2 (IL-2), interleukin-12(IL-12) or combinations thereof. Cells that produce suppressivecytokines can also be detected. Examples of suppressive cytokinesinclude but are not limited to interleukin 10 (IL-10), scurfin (FOXP3gene product), transforming growth factor beta (TGF-beta), CTLA4, andcombinations thereof. The presence, absence, amount or relativeexpression of any of the proteins described above and below, as well asother predetermined proteins can be measured.

Many of the measurements are made by taking advantage of proteinbiomarkers on the cells that indicate the above described parameters.Biomarkers useful in the inventive method include: CD3 (T-cell), CD4(T-helper), CD8 (T-cytotoxic), CD19 (B-cell), CD11c (type 1 dendriticcell), CD123 (type 2 dendritic cell), CD14 (monocyte), CD45RO (memory),CD45RA (naïve), CD25high (highly activated), CD25low (activated),CD25negative (inactive), CD154 (antigen-specific activation), CSFElow(proliferative), annexin V (apoptotic), 7AAD (dead), CD27 (characterizesmemory), CD28 (senescence), CD62 ligand (lymph node homing receptor),CD86 (costimulatory molecule), CD 69 (activation marker)), CD54(costimulatory molecule), CD95 (fas ligand), CD71 (transferrinreceptor), PD-1 (costimulatory molecule), PD1L (costimulatory molecule),ICOS (costimulatory molecule), CCR4 (chemokine receptor), CCR5(chemokine receptor), CCR7 (chemokine receptor), CD16/56 (NK cell), ILT3(inhibitory marker on antigen-presenting cells), HLA-DR, andcombinations thereof. For example, markers that are specific to cellfunction include annexin V (apoptosis), active caspases, 7-AAD (death),CD25 (activation marker), CD69 (activation marker), CD71 (activationmarker), CD86 (activation marker), and CD54 (activation marker).Examples of markers specific to cell differentiation include CD45RO,CD45RA, CD27, CD62 ligand, CD28, CCR4 (chemokine receptor), CCR5(chemokine receptor), CD34, CD134, CD27, HLA-DR, CD11c, CD123, CTLA4,CD3, CD4, CD8, CD16/56, CD19, CD14, and ILT3.

In order to measure the above described physical and functionalattributes, especially the presence and/or quantity of proteins, markerscan be utilized to label the cells to be analyzed. Such markerstypically are colored dyes that are able to be detected anddifferentiated by the flow cytometer. Marker dyes that persist in thecell can be used to distinguish one cell type from another in a mixedcell population. Examples of markers suitable for use with the presentinvention include: carboxyfluorescein diacetate succimidyl ester (CFSE,Molecular Probes, Eugene, Oreg.), EMA (cell viability dye), 7-AAD (cellviability dye) and Quantum dots, such as having emission spectra between545 nm and 800 nm (Quantum Dot Corp., Hayward, Calif.). The use of suchdyes is known in the art, and includes, for example, conjugating them toantibodies or other ligands for a specific protein. Compositionscomprising these markers are also contemplated within the invention andcan be used with the inventive method.

While any suitable equipment and methodology for measuring the multipleparameters can be employed, the preferred platform for measuring themultiple parameters is a flow cytometer. Flow cytometers capable ofdetecting and differentiating at least 4 (and more preferably at least7) differently colored markers are preferred. More preferred is a flowcytometer capable of detecting and differentiating at least 10 and morepreferably at least 19 or at least 25 differently colored markers. Infact, depending on the number of lasers in a flow cytometer, theinventive method is able to measure and compare in excess of 50 multipleparameters, such as in excess of 75 multiple parameters or even over 100multiple parameters. The number of parameters which the inventive methodcan detect is limited by the number of lasers and detectors in suchsystems. In one embodiment, up to 240 lymphocyte subfamilies can becharacterized simultaneously for the patient and test sample in eachreaction mixture. Methods of using a flow cytometric machine arepublicly available and are known to those in the art. Of course, whileflow cytometers are preferred for use in the context of the presentinvention, some parameters can be measured using other methodology. Forexample, migratory behavior or cellular aggregation can be assessedusing cell counters, microscopy, and other techniques.

In accordance with the inventive method, multiple parameters aremeasured in the reactions of the cells in the test reaction and thethird party reaction. The unreacted third sample from the patient servesas a control. Desirably, the parameters are quantified to permitrelative values to be determined between the test reaction and the thirdparty reaction. Thus, for each parameter, the patient-test subjectreaction can be expressed as a fraction (ratio) of the patient-thirdparty reaction, and the battery of multiple ratios can then serve as abasis for addressing the relevance of the reaction to the desiredclinical event. Alternatively, the inventive method may be utilized togauge the test subject's reaction to the patient's sample versus thetest subject's reaction to the third party. This is made possible bysimply reversing the comparison of the parameters, that is, comparingthe test subject's sample to the patient's sample and the third party'ssample, and allows a determination of whether the test subject's (e.g.,a donor) cells are immunologically reactive to the patient's cells.Therefore the inventive method provides a means of assessing thepatient's immunological status on both sides of the reaction, that is,the patient's reaction to the donor's cells, and the donor's reaction tothe patient's cells. Thus, the method can be used to assessimmunomodulation of the patient vis-à-vis the donor and also the graftvis-à-vis the patient recipient, e.g., to gauge the risk of rejectionand also the development of tolerance.

In one embodiment, the clinical event is the titration of a patient'simmunosuppression. The titration of immunosuppression can be after organtransplantation, or during a viral or bacterial infection. Further, thetitration can be during a viral or bacterial infection after a patienthas undergone organ transplantation. In another embodiment the clinicalevent can be the comparison of one or more immunosuppressive regimens inwhich the patient is undergoing a first immunosuppressive regimen andanother patient or subject is undergoing a second immunosuppressiveregimen and the third subject or patient is not undergoing animmunosuppressive regimen.

In another embodiment, the clinical event can be monitoring or assessingthe risk of organ rejection, the event of organ rejection, the degree orseverity of organ rejection, or graft dysfunction in suspectedrejection. In yet another embodiment, the clinical event can bemonitoring the severity of one or more bacterial or viral infections.Further, the clinical event can include monitoring the response of apatient to immunosuppressive agents, (such as FK-506) the withdrawal ofan immunosuppressive agent, an antiviral agent, or an anti-bacterialagent.

When the patient is an organ transplant recipient, the organ can be bonemarrow or a solid organ. Examples of solid organs include liver,intestine, kidney, heart, lung, pancreas, and sections and combinationsthereof.

When the patient's response to an infectious agent is being monitored,the infectious agent can be one or more bacteria and/or viruses. Virusesthat elicit an immune response or suppress immune response arecontemplated, and include Epstein-Barr, adenovirus, and cytomegalovirus.Additional infectious agents include bioterrorism agents such asanthrax, botulism, etc.

In yet another embodiment, the patient's immune response is assessed bycomparing multiple parameters in the reaction of the patient's samplethat has been exposed to the test sample (the test reaction), with thepatient's sample exposed to the third party (the third party reaction),in order to determine the patient's immune status. In general, if thepatient's sample is more reactive to the test sample than the thirdparty sample, an increased risk of rejection, in a transplant patient,is likely. In contrast, if the patient's sample is less reactive to thetest sample than the third party sample then a reduced risk of rejectionis likely. In another example, a greater degree of proliferation in ahighly activated memory phenotype of T-helper cells within the testreaction compared to that of the third party reaction can indicate aheightened risk of rejection in an organ transplant patient. In anotherexample, a lower degree of proliferation in a highly activated memoryphenotype of T-helper cells within the test reaction compared to that ofthe third party reaction can indicate a lower risk of rejection in anorgan transplant patient. In another example, a greater number ofpro-inflammatory-cytokine-producing cells and lesssuppressive-cytokine-producing cells within the test reaction than thatof the third party reaction can indicate a heightened risk of rejectionin an organ transplant patient. Alternatively, a lesser number ofpro-inflammatory-cytokine-producing cells and more suppressive-cytokineproducing cells within the test reaction compared to that of the thirdparty reaction can indicate a reduced risk of rejection in an organtransplant patient.

Additionally, many cytokines respond in a dose-dependent fashion toanti-rejection drugs. Therefore, a clinician can utilize the patient'sown internal targets (these responsive cytokines) to establish athreshold of treatment (i.e., the ¼, ½, and ¾-maximal inhibition) uniqueto the patient. That is, the method allows therapy to be tailored to thepatient and the endpoint of therapy to be determined.

The comparison of reactions is not limited to the ratio of test subjectversus third party. For instance precursor frequencies of reactive cellsin the parent population, or the absolute numbers of cells of a givensubtype, or an absolute number of cells of a given subtype expressed asa fraction of the absolute number of cells of another subtype can bedetermined. For example, when precursor frequencies are measured, if theprecursor frequencies of the test subject-reactive subpopulations,classified by multiple parameters, exceeds a threshold value it canindicate an enhanced risk of rejection. Alternatively, if the precursorfrequencies of the test subject-reactive subpopulations, classified bymultiple parameters, falls below a threshold value, it can indicate areduced risk of organ rejection in a transplant patient.

In another embodiment, the inventive method allows multiple parametricmeasurements of the patient's cell populations at different time points.For example, measurements can be made before, during and after animmunosuppressive regimen, before and after treatment with ananti-infectious agent, or alternatively, before and then after organtransplantation. For example, multiple parametric measurements of thepatient's cell subpopulations can be measured at different time pointsbetween two or more consecutive doses of an anti-rejection drug. Themeasurements can be used to define the upper (safe) and lower(effective) limits of the anti-rejection drug therapy for that patient.In this way, an immunosuppressive dosing regimen can be tailored to thepatient taking into account his or her unique and dynamic host responseto the transplanted organ as well as to the immunosuppressive agents.For instance, suppression of a patient's immune system can be titratedutilizing an immunoreactivity index, such as mixed lymphocyte reaction(MLR). Essentially, the information can be interpreted such that if theratio of the test subject to third party reactivity is greater than 1,then the FK level, that is the dose of FK-506 (i.e., PROGRAF®, AstellasPharma US Inc.) required by the patient for adequate immunosuppression,is likely to be above 10 ng/ml, such as up to 20 ng/ml. If the ratio isless than 1, then the FK level is likely to be in the 7-8 ng/ml range.

Additionally, the inventive method can be utilized to determine whetherthe response to drug therapy indicates resolution of rejection risk. Itcan also be used to test whether the reduction of drug therapy increasesthe risk of rejection and whether drug therapy, if discontinued, shouldbe resumed. This provides a powerful tool for the clinician such thatover-medication and under-medication of a given patient can be avoidedand duration of treatment can be tailored to the needs of the patient.Therefore the clinician would no longer have to rely on general dosesand time courses that tend to work in the greater transplant population.

In another embodiment, the effect of the patient's immune system on thetest subject's immune system can be determined. This is possible becausethe invention provides a method for assessing the status of the cellpopulations in each sample, that is the patient's sample, the testsample, and the third party sample, both before and after they have beenreacted together. For example, if the donor cell populations areinhibited by the patient's cells, then the patients' immune system hasdeveloped regulatory or immunomodulatory capabilities, that is,anti-rejection properties. Patients who have achieved a state of“tolerance” require less anti-rejection medication and thereforeknowledge of this state is particularly useful when treating thesepatients. Conversely, if no such anti-rejection property has beenachieved, the patient has not developed tolerance and therefore thatpatient's anti-rejection therapy should not be reduced.

As noted above, the power of the inventive method is limited by thenumber of lasers and detectors available to conduct flow cytometry.However, in the event that one is interested only in a small fraction ofthe utility of the assay, then a small part of the assay can suffice.For example, equipped with only a 4-color flow cytometer, it is stillpossible to detect rejection risk by using 4 parameters—one each forrecipient CD4, recipient memory (CD45RO), and recipient intravitallabeling (CFSE). The donor and third party stimulator cell can beexcluded by labeling with a marker that is present on all types of PBL(CD45). However, the assay would require a 3-4 day incubation.Increasing the number of parameters permits a more rapid detection. Forexample, using a 5- or 6-color flow cytometer, CD25 can be added as aparameter. In this case, the recipient CD4+CD45RO+CD25+ cell canproliferate rapidly in response to the donor or third-party cell(labeled with CD45). Such an assay can show donor-specific proliferationwithin 1-2 days.

In another embodiment, the invention provides a method of assaying theimmune response of a test subject (or patient) to a viral antigen. Inaccordance with the method, an antigen derived from a virus-of-interestis labeled with a dye-conjugated MHC-tetramer. Also, a tissue samplecomprising CD8+ cells is obtained from the test subject, and CD8+ withinthe sample are exposed to the labeled viral antigen. Thereafter, thecells are assayed to identify any CD8+ cells that also carry the dye towhich the MHC-tetramer is conjugated. Typically, this is achieved byflow cytometry, using one dye that labels CD8 and the dye-conjugatedMHC-tetramer. The presence of such dually-reactive cells indicates thatthe immune system of the patient is responsive to the viral antigen. Themethod can serve as a basis for identifying individuals at risk forcontracting an infection from such virus (by the absence ofdually-reactive CD8+ cells) and those who can be expected to mount aneffective immune response if challenged by the virus.

In another embodiment, the invention provides a method of assayingdonor-specific alloreactivity, which can be use to assess a testsubject's (recipient's) risk of rejection of tissue from a donor. Inaccordance with this aspect of the invention, the method comprises firstobtaining a tissue sample from the test subject (recipient) and exposingcells within the tissue sample to class I and class II MHC antigens.Thereafter, the lymphocytes from the test subject are assayed to assesswhether they have become activated as a result of exposure to the MHC Iand MHC II antigens (for example, by screening for presence of CD154,CD95, and/or CD71). Activation of the cells from the test subject isindicative that the test subject is at risk of rejecting tissue form thedonor. This approach requires knowledge of recipient and donor HLAantigens (tissue typing) in much the same manner as the cell-cell mixedlymphocyte response. The difference is that one has to pick commerciallyavailable/or custom synthesized MHC peptides from a commercial or customlibrary.

EXAMPLE 1

The examples further illustrate the invention but, of course, should notbe construed as in any way limiting its scope.

This example demonstrates the rapid detection of donor-specificalloreactivity with highly activated memory T-helper cells in CFSE-MLRwith 7-color flow cytometry.

The participating subjects were sixteen liver transplant patients (LTx),ages ranging from 0.45-18 years, with thymoglobulin (5 mg/kg) inductionand undergoing Tacrolimus monotherapy. Peripheral lymphocytes (PBL) fromeach subject were stained with 1 μM carboxyfluorescein diacetatesuccimidyl ester (CFSE), and incubated for 24 hours with irradiateddonor (HLA-DR matched) and third-party (HLA-DR unmatched) PBL at a 1:1ratio. Frequencies of naïve (CDR45RA+), memory (CDR45RO+) anddouble-positive (CD45RA+RO+) CD4+, CD8+, and CD19+ cells expressing theactivation marker CD25 to varying degrees (CD25hi, CD25lo, CD25neg) weremeasured by 7-color flow cytometry. Each lymphocyte subset (CD4+, CD8+,and CD19+) was thus divided into 9 subpopulations. For each of the 27subphenotypes (e.g. CD4+25hiRO+), cells demonstrating >3-fold dilutionof CFSE in daughter cells, due to activation-induced proliferation, weremeasured. The immunoreactivity index (IR) for each phenotype wascalculated as the multiple by which donor-induced proliferation exceededthat due to third-party. This was correlated with a similar indexdetermined by 5-day, 3H-thymidine MLR (mixed lymphocyte reaction). AnIR>1 reflected increased rejection risk. An IR<1 reflected decreasedrejection risk. An IR derived from both types of MLR was correlated.

The study found 8 non-rejecting subjects, 7 rejecting subjects, and onesubject removed from immunosuppression due to PTLD (post-transplantlymphoproliferative disorder). The IR calculated by the proliferativeresponse of CD4+hiCD45RO-cells in CFSE-MLR correlated well with the IRcalculated by 5-day MLR (regression (r2)=0.60). The correlation betweenthe remaining 26 subpopulations of T- and B-cells evaluated in CFSE-MLRand 5-day MLR was poor (r2 values ranging from 0.004 to 0.21). The 5-dayMLR demonstrated IR<1 in 7 of the 8 non-rejecting subjects, the subjectwith PTLD, and 2 of the 7 rejecting subjects. CFSE-MLR based onCD4+CD25hiCD45RO+ cells indicated increased rejection risk (IR>1) in 6of the 7 rejecting subjects after the rejecting episode and in 2 of the8 non-rejecting subjects during the first 60 days after LTx. The subjectwith PTLD and 6 of the 8 non-rejecting subjects demonstrated an IR<1 inCFSE-MLR (CD4+CD25hiCD45RO+).

The study demonstrates that memory T-helper cells, which express highamounts of the activation marker CD25 can express donor-specificalloreactivity more rapidly, and as effectively as classical3H-thymidine MLR, which can facilitate rapid identification of rejectionrisk and safer strategies to minimize clinical immunosuppression.

EXAMPLE 2

The example demonstrates donor-specific immunoreactivity can be measuredrapidly by proliferation of activated, memory T-helper cells in flowcytometric, CFSE-MLR.

To evaluate the proliferation of CFSE-labeled recipient T-helper (TH)subsets, co-cultured with HLA-DR matched (donor, D) and mismatched(third-party, TP) lymphocytes (PBMC) in subjects with primary livertransplants were evaluated. Thirty LTx recipients, ages 0-18 years, werepretreated with 5 mg/kg thymoglobulin, and steroid-free Tacrolimusmonotherapy. Naïve (CD45RA+) or memory (CD45RO+) CD4+ cells, with orwithout an activation marker (CD25hi, CD25lo, CD25neg) were enumeratedby flow cytometry pre-LTx, and at 1, 3, 6, and 12 months post-LTx in 14non-rejecting subjects and in 16 rejecting subjects. Thereafter,TH-proliferation was measured by CFSE-dilution using 5-color flowcytometry in 10/30 recipients, after 24-hour co-culture with D- andTP-PBMC. Subpopulations demonstrating ≥3-fold dilution of CFSE were usedto calculate immunoreactivity to donor relative to TP (D-THproliferation: TP-TH proliferation) in TH subsets.

Results of classical MLR with 3H-thymidine incorporation after 5-dayco-culture were expressed as the ratio of Donor Stimulation Index (DSI):TPSI and correlated with immunoreactivity measured by CFSE dilution. Asignificant depletion in CD24hiCd45RO+TH cells in the first 6 monthsafter LTx in rejecting subjects was seen (Mean absolute counts per mm3:Pre-LTx was 62 vs. 24 in month 1 (p=0.047), vs. 6 in month 2 (p=0.01),vs. 17 in months 4-6 (p=0.35)). A significant depletion was also seen inabsolute counts of both naïve and memory TH cells that were CD25neg inrejecting subjects. Despite a numeric decrease from pre-LTx counts, suchdifferences were not statistically significant in non-rejectingsubjects. Good correlations were seen between DSI:TPSI ratio measured byMLR and Donor:TP TH cell proliferation by CFSE-dilution in memory THcells which were CD25hi, CD25lo, and CD25neg (r2=0.72, 0.91, and 0.88respectively). Furthermore, donor-specific immunoreactivity measured byMLR and CFSE-proliferation of CD25hiMemoryTH cells, exceededimmunoreactivity to TP in rejecting subjects, but was less than TP innon-rejecting subjects.

This study demonstrates that memory TH cells expressing high amounts ofthe activation marker CD25 can represent a rapid and sensitive measureof donor-specific immunoreactivity, which could be used to titrateimmunosuppression to the rejection risk in a given patient.

EXAMPLE 3

This example demonstrates the relationship of immunosuppressive druglevels to MLR results.

In this study, 37 children receiving thymoglobulin, andTacrolimus/Sirolimus (TAC/SRL) monotherapy were observed. Serialmeasurements tested whether resolution of the risk of acute cellularrejection (ACR-risk) could be defined by (median±SEM) time to achieve:(1) Normal Graft function (SGOT/SGPT<40 IU/ml, Tbili<2 mg/di), (2) “Low”immunosuppression (TAC/SRL level<8 ng/ml), and (3) Immunoreactivityindex (IR)<1 in 3H-thymidine MLR. This was the multiple by whichrecipient response to donor (HLA-DR-matched) lymphocytes exceededresponse to third-party (HLA-DR-mismatched). An IR>1 reflected increase,while IR<1 reflected decreased ACR-risk. The median age was 4 years(range 0.45-18 years), and follow-up 570 days (range 106-1144). 20subjects experienced biopsy-proven early ACR and 17 did not. Comparedwith non-rejecting subjects, rejecting subjects demonstratedsignificantly greater (p<0.05, Kaplan-Meier) median time to achieveIR<1, TAC/SRL<8 ng/ml and significantly more recurrent/delayed ACR (35%vs 6%, p=0.032). No such differences occurred for graft function. Also,SEM values suggested a 4-fold greater variation in median time todecreased ACR-risk (IR<1) in rejecting subjects compared withnon-rejecting subjects. TAC/SRL levels were 4±0.6 ng/ml at recurrence,due to clinical misjudgment (n=6) and non-compliance (n=2). In tworejecting subjects, recurrence was associated with IR 1.1 and 2.1.

This study demonstrates that during immunosuppression minimization inchildren, the risk of liver graft rejection can be related to increasedimmunosuppression requirements and persistent donor-specificimmunoreactivity, but not to graft function. Serial monitoring ofimmunoreactivity can reduce this risk, especially among rejectingsubjects.

EXAMPLE 4

This example demonstrates that memory Th proliferative responses can beused to measure rejection risk.

Ten T-cell sub-phenotypes have been evaluated for their ability tomeasure the instantaneous risk of rejection in 43 children with liverand small bowel transplantation (LTx, SBTx).

Methods: The subject population included 39 immunosupressed childrenwith LTx (n=28) or small bowel transplantation (SBTx, n=15) all of whomhad received rabbit anti-human thymocyte globulin and steroid-freeTacrolimus. Also included were 4 children with LTx, in whom therejection-free course was maintained without any immunosuppression(tolerant). The assay system comprises mixed lymphocyte coculture, inwhich the indicator of proliferation is dilution of the intravital dye,carboxyflouresciensuccinimydyl ester (CFSE).

Recipient peripheral blood lymphocytes (PBL) which have been purified byFicoll gradient, are incubated alone, or with PBL from donor orthird-party, for a period of 1-4 days, at a ratio of 1:1. The incubationconditions include 37 degrees Centigrade, routine culture medium with5-10% fetal calf serum, and 5% carbon dioxide. Recipient PBLproliferation is measured in the parent T-helper (Th, CD4+) andT-cytotoxic (Tc, CD8+) cells, and their memory, naïve, activated, andactivated-memory subphenotypes using multicolor flow cytometry. Examplesof markers for each of these subphenotypes are CD45RO+(memory), naive(CD45RO- or CD45RA-), activated (CD25+) and activated memory(CD45RO+CD25+). The youngest generations of proliferating CFSE-labeledrecipient PBL constitute the indicator of alloreactivity. This indicatoris measured in each subphenotype. Dead cells are excluded with 7-AADlabeling. Pre-labeling of stimulator donor and third-party cells byanti-CD45-APC or anti-CD45-Pacific Blue prevents admixture withresponder cells during analysis. The risk of rejection is equated withdonor PBL-induced proliferation, but expressed as a fraction ofsimultaneously measured, third-party-induced proliferation of recipientPBL. This type of indexing is done to minimize intrapatient variabilitythat may arise from non-alloantigenic stimuli, because such stimuli havean equal likelihood of influencing both, donor-specific and third-partyalloresponses on any given day. The resulting immunoreactivity indexreflects a higher risk of rejection if >1 and a lower risk of rejectionif <1.

Results (Tables 1-2): Immunosupressed children experiencing allograftrejection within the first 60 days after transplantation (n=24)demonstrated a significantly higher immunoreactivity index in memoryT-helper cells, when compared with children who have been rejection-freeon maintenance immunosuppressants (n=15, 1.29±0.8 vs 0.5±0.22, p=0.0012,corrected for simultaneous measurement of 10 subphenotypes).Significantly higher immunoreactivity index in memory T-helper cells wasalso seen when rejectors (immunosuppressed) were compared with tolerantchildren (1.29±0.8 vs 0.421.29±0.80.11, corrected p=0.00034). Parent Thand Tc cells demonstrate borderline significance, which disappears aftercorrection for multiple testing (Tables 1 and 2). Naïve Th cells appearto be significantly more hyporesponsive to donor-alloantigen amongtolerant patients, when compared with non-rejectors who areimmunosuppressed (immunoreactivity index 0.28±0.12 vs 0.68±0.29,corrected p=0.015).

CONCLUSIONS

Together, these observations support the use of memory Th proliferativeresponses in measuring instantaneous rejection risk in transplantrecipients. If measured longitudinally in individual recipients, thesimultaneous evaluation of immunoreactivity indices within memory andnaïve Th cells may improve the safety with which immunosuppressants areminimized or eliminated after transplantation.

TABLE 1 Immunoreactivity indices for CD₄+ cell and its subphenotypes inLTx children. CD₄+ Activated- Parent Memory Memory Naïve ActivatedRejectors (R) 1.18 ± 0.92 1.29 ± 0.81 1.11 ± 0.91  2.6 ± 6.29 3.33 ±5.7  Non-Rejectors (NR) 0.85 ± 0.24  0.5 ± 0.22 0.59 ± 0.34 0.68 ± 0.290.73 ± 0.25 Tolerant (TOL) 0.73 ± 0.18 0.42 ± 0.11  1.2 ± 0.82 0.28 ±0.12 1.33 ± 1.14 R vs NR p value 0.119 0.000119 0.02699 0.1485310.069776 corrected 1 0.001194 0.269897 1 0.697763 R vs TOL p value0.32218874 3.4E−05 0.855689 0.08491 0.197708 corrected 1 0.00034 10.849102 1 NR vs p value 0.32218874 0.371333 0.241982 0.001517 0.457012TOL corrected 1 1 1 0.015171 1

TABLE 2 Immunoreactivity indices for CD₃+ CD₄− (T-cytotoxic/CD8+) celland subphenotypes in LTx children. CD8+ Activated- Parent Memory MemoryNaïve Activated Rejectors(R) 1.91 ± 1.7  1.77 ± 1.49 1.54 ± 1.73 1.49 ±1.83 1.55 ± 1.32 Non-Rejectors (NR) 0.92 ± 0.43 0.83 ± 0.47 0.93 ± 0.6  0.7 ± 0.27 0.82 ± 0.58 Tolerant (TOL) 0.73 ± 0.3  0.56 ± 0.09 1.28 ±0.78 0.73 ± 0.79 0.48 ± 0.45 R vs NR p value 0.03311915 0.033734 0.21770.104818 0.089756 corrected 0.33119154 0.337342 1 1 0.897556 R vs TOL pvalue 0.01494483 0.007425 0.691887 0.230728 0.078566 corrected0.14944834 0.074249 1 1 0.785658 NR vs p value 0.36166837 0.0701620.532643 0.934819 0.447136 TOL corrected 1 0.701618 1 1 1

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments can become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

I claim:
 1. A method of assessing the reaction of a patient's immunesystem to an antigen stimulant, comprising: contacting a first samplecomprising T-cells obtained from the patient with a viable antigen underconditions sufficient to induce T cell proliferation by the viableantigen; contacting a second sample comprising T-cells obtained from thepatient with a viable third-party antigen under conditions sufficient toinduce T- cell proliferation by the viable third-party antigen;measuring T cell proliferation induced by the viable antigen in thefirst sample by detecting a plurality of markers in the first samplefollowing contacting the first sample with the viable antigen, and atleast one additional marker, respectively, wherein the plurality ofmakers comprises CD154, CD45RO and CD8; measuring T cell proliferationinduced by the viable third party antigen in the second sample bydetecting the plurality of markers in the second sample followingcontacting the second sample with the third-party antigen, and the atleast one additional marker, respectively; and comparing T cellproliferation induced by the viable antigen in the first sample to Tcell proliferation or an immune response induced by the viablethird-party antigen in the second sample, thereby determining that thepatient's immune system responds to the viable third party antigen andthat the viable third party antigen induces reaction of the patient'simmune system.
 2. The method of claim 1, wherein the additional markeris PD-1, PD-L1, ICOS, or IFN-gamma.
 3. The method of claim 2, furthercomprising assessing viability of T-cells in the first sample andviability of T-cells in the second sample.
 4. The method of claim 3,wherein assessing viability of T-cells comprises use of a cell viabilitydye.
 5. The method of claim 4, wherein the cell viability dye isethidium monoazide (EMA) or 7-amino-actinomycin D (7-AAD).
 6. The methodof claim 1, wherein the first sample and the second sample areperipheral blood lymphocytes or peripheral blood leukocytes.
 7. Themethod of claim 1, wherein the patient is a human.
 8. The method ofclaim 7, wherein the patient is an adult.
 9. The method of claim 7,wherein the patient is a child.
 10. The method of claim 1, wherein theplurality of markers further comprises one or more of CD45RA, CD27,CD62, CD28, CCR4, CCR5, CD34, CD134, HLA-DR, CD11c, CD123, CD3, CD4,CD16/56, CD19, CD14, ILT3, CD25, 7-AAD, CD69, CD71, CD86, IL-2, TNFalpha, CFSE, TGF-beta, Annexin V, CFSE, a caspase, IL10, IL-12, IL-2,CCR7, CD56, and CD54.
 11. The method of claim 1, wherein the viableantigen or the viable third party antigen comprise an antigenic peptide,an antigenic peptide labeled with a flourochrome, or a combinationthereof.
 12. The method of claim 1, wherein the T-cells are T-helper orT-cytotoxic lymphocytes.
 13. The method of claim 1, wherein the firstsample and the second sample are further contacted with animmunomodulatory drug.
 14. The method of claim 13, wherein theimmunomodulatory drug is an anti-rejection agent.
 15. The method ofclaim 1, wherein the viable antigen is an autoantigen, viral peptide,bacterial peptide, antigen attached to tetramer, or an antigenic peptideattached to a monomeric protein, polymeric protein or peptide.
 16. Themethod of claim 1, wherein the the at least one additional markeris-CTLA4, IFN-gamma.
 17. The method of claim 1, wherein the at least oneadditional marker is PD-1 or PD-L1.
 18. The method of claim 1, whereinthe at least one additional marker is IFN-gamma.